P0144Targeting FTO in preadipocytes inhibits creeping fat formation and MMP9-mediated fistulogenesis in Crohn’s disease

Background Matrix metalloproteinase-9 (MMP9) contributes to fistula pathogenesis by degrading extracellular matrix, and dysfunctional preadipocytes have also been associated with this process. However, how preadipocytes regulate MMP9-mediated fistulogenesis are not fully understood. This study aims to elucidate the pathogenic mechanisms by which preadipocytes contribute to fistula development in Crohn’s disease (CD), thereby providing insight for future therapeutic interventions. Methods Single-cell RNA sequencing (scRNA-seq) was performed on clinical intestinal tissues to characterize transcriptional profiles of CD-associated fistulas, especially adipocyte subpopulation. Ftoflox/flox; Fabp4-Cre mice were generated and studied in a colitis with intestinal fistula model. The effect of FTO knockdown in preadipocytes on MMP9-mediated fistulogenesis was assessed via qRT-PCR, Western blot, CCK-8, ELISA, Oil Red O staining, and flow cytometry. RNA-seq and SRAMP database identified the mRNA carrying m6A modifications, and high-confidence m6A sites were confirmed by MeRIP-qPCR. Results Single-cell transcriptomic analysis revealed that MMPs was highly expressed in CD-associated fistulas compared to normal controls, while FTO was significantly upregulated in adipocytes of creeping fat (CF). In a TNBS-induced intestinal fistula model, Ftoflox/flox; Fabp4-Cre mice demonstrated accelerated fistula healing by suppressing MMP9 expression compared to Ftoflox/flox mice. In vitro assays indicated that targeting FTO in preadipocytes downregulates ACSL3 through enhanced m6A modification, inducing ROS-dependent ferroptosis that subsequently reduces MMP9 expression via inhibition of the JNK–c-Jun pathway, ultimately attenuating fistula formation. The m6A modification sites on ACSL3 mRNA were identified within the motifs at nucleotide regions 1040–1216, 2414–2555, and 2794–2905. Conclusion Targeting FTO in preadipocytes inhibits MMP9 expression via ferroptosis and also regulates m6A methylation of ACSL3. The study highlights the significance of modulating FTO and MMP9 expression in preadipocytes of creeping fat, laying a theoretical foundation for therapeutic strategies against creeping fat and CD-associated fistulas. Conflict of interest: Chen, Jiayue: No conflict of interest Prof. Zhao, Xinmei: No conflict of interest